Article reSeArcH
COOT. Similarly, the crystal structure of Ca^2 +-CaM in complex with the RyR1
peptide (PDB code 2BCX) was fitted into the maps obtained in the presence of
Ca^2 +-CaM and manually adjusted in COOT. Structure refinement was performed
using PHENIX^59 in real space with restrained secondary structure and geometry.
The statistics of the three-dimensional reconstruction and model refinement are
summarized in Extended Data Table 1.
Evaluation of the conformations of N- and C-lobes of CaM. Different conforma-
tions of N- and C-lobes were docked into the electron microscopy reconstruction
for FKBP12.6/ATP/caffeine/high-[Ca^2 +]/Ca^2 +-CaM using the ‘Fit in Map’ tool of
Chimera, selecting the options that include ‘Real-time correlation’, ‘7-Å resolution
of Use map simulated from atoms’, ‘Use only data above contour level from first
map’, ‘Optimize correlation’, ‘Correlation calculated about mean data value’, ‘Allow
rotation and shift’ and ‘Move whole molecules’.
Site-directed mutagenesis. Point mutations in mouse Ryr2 and in human CALM1
were generated with the overlap extension method using PCR. In brief, a EcoRV/
HpaI DNA fragment containing the RyR2(Y2156A) mutation and an AgeI/SalI
fragment containing the RyR2(V3599A), RyR2(W3587A) or RyR2(L3590A) muta-
tions were obtained by overlapping PCR and used to replace the corresponding
wild-type fragment in the NheI/BsiWI fragment of Ryr2, The mutated NheI/BsiWI
fragment was then used to replace the corresponding wild-type fragment in the
full-length Ryr2 cDNA in pcDNA5. A HindIII/XhoI full-length CALM1 DNA
fragment containing various point mutations was generated by overlapping PCR,
which was then subcloned into pcDNA3. All point mutations in Ryr2 and CALM1
were confirmed by DNA sequencing.
Generation of stable, inducible cell lines expressing RyR2(WT) and mutants.
Stable, inducible HEK293 cell lines expressing RyR2(WT), RyR2(Y2156A),
RyR2(V3599A), RyR2(W3587A) and RyR2(L3590A) were generated using the
Flp-In T-REx Core Kit from Invitrogen. These cell lines were not authenticated.
These cells tested negative for mycoplasma contamination. In brief, Flp-In T-REx
HEK293 cells were co-transfected with the inducible expression vector pcDNA5/
FRT/TO containing the Ryr2WT or Ryr2-mutant cDNA and the pOG44 vector
encoding the Flp recombinase in 1:5 ratios using the calcium phosphate precipi-
tation method. The transfected cells were washed with phosphate buffered saline
(PBS; 137 mM NaCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 and 2.7 mM KCl, pH 7.4)
24 h after transfection followed by a change into fresh medium for 24 h. The cells
were then washed again with PBS, collected and plated onto new dishes. After the
cells had attached (around 4 h), the growth medium was replaced with a selection
medium containing 200 μg ml−^1 hygromycin (Invitrogen). The selection medium
was changed every 3–4 days until the desired number of cells was grown. The
hygromycin-resistant cells were pooled, aliquoted (1 ml) and stored at −80 °C.
These positive cells are believed to be isogenic, because the integration of Ryr2
cDNA is mediated by the Flp recombinase at a single FRT site.
Single-cell luminal Ca^2 + imaging. Luminal Ca^2 + levels in RyR2WT- or Ryr2-
mutant-expressing HEK293 cells transfected with or without CaM(WT) or CaM
mutants were measured using single-cell Ca^2 + imaging and the fluorescence
resonance energy transfer (FRET)-based endoplasmic-reticulum luminal Ca^2 +-
sensitive chameleon protein D1ER as previously described^60 ,^61. The cells were
grown to 95% confluence in a 75-cm^2 flask, dissociated with PBS (137 mM NaCl,
8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 and 2.7 mM KCl, pH 7.4) and plated on glass
coverslips placed on tissue culture dishes at approximately 10% confluence 18–20
h before transfection with cDNA for D1ER and cDNAs for CaM(WT) or CaM
mutants using the calcium phosphate precipitation method. After transfection for
24 h, the growth medium was then changed to an induction medium containing 1
μg ml−^1 tetracycline. After induction for around 22 h, the coverslip was mounted
onto an inverted microscope (Nikon TE2000-S) and the cells on the coverslip were
perfused continuously with Krebs–Ringer–HEPES buffer (125 mM NaCl, 5 mM
KCl, 1.2 mM KH 2 PO 4 , 6 mM glucose, 1.2 mM MgCl 2 and 25 mM HEPES, pH 7.4)
containing various concentrations of CaCl 2 (0, 1 and 2 mM) to induce SOICR,
followed by the addition of 1.0 mM tetracaine, which was used to estimate the store
capacity, and caffeine (20 mM), which was used to estimate the minimum store level
by depleting the endoplasmic-reticulum Ca^2 + stores at room temperature (23 °C).
Images were captured with Compix Simple PCI 6 software every 2 s using the Nikon
TE2000-S inverted microscope equipped with an S-Fluor 20×/0.75 NA objective.
The filters used for D1ER imaging were λex = 436 ± 20 nm for CFP and λex =
500 ± 20 nm for YFP, and λem = 465 ± 30 nm for CFP and λem = 535 ± 30 nm
for YFP with a dichroic mirror (500 nm). The amount of FRET in individual cells
was determined from the ratio of the light emission at 535 and 465 nm. FSOICR
is defined as the FRET level at which SOICR occurs, and Ftermi is defined as the
FRET level at which SOICR terminates. The maximum FRET signal Fmax is defined
as the FRET level after tetracaine treatment. The minimum FRET signal Fmin is
defined as the FRET level after caffeine treatment. The termination and activa-
tion thresholds of SOICR in individual cells were determined using the equations
shown in Extended Data Fig. 7a. The store capacity is calculated by subtracting Fmin
from Fmax. Individual data points represent the average measurements of around
10–30 cells from one coverslip in one set of experiment. The number of experi-
ments and coverslips for each condition is used as the sample size for data analyses.
Single-channel recordings in planar lipid bilayers. Recombinant RyR2(WT) and
mutant channels were purified from cell lysates prepared from HEK293 cells trans-
fected with the Ryr2WT or Ryr2-mutant (Y2156A, V3599A, W3587A or L3590A)
cDNA by sucrose density gradient centrifugation as previously described^62. Heart
phosphatidylethanolamine (50%) and brain phosphatidylserine (50%) (Avanti
Polar Lipids), dissolved in chloroform, were combined and dried under nitrogen
gas and resuspended in 30 μl of n-decane at a concentration of 12 mg lipid per ml.
Bilayers were formed across a 250-μm hole in a Delrin partition separating two
chambers. The trans chamber (800 μl) was connected to the head stage input of an
Axopatch 200A amplifier (Axon Instruments). The cis chamber (1.2 ml) was held
at virtual ground. A symmetrical solution containing 250 mM KCl and 25 mM
HEPES, pH 7.4 was used for all recordings, unless indicated otherwise. A 4-μl
aliquot (around 1 μg protein) of the sucrose density gradient-purified recombinant
RyR2(WT) or mutant channels was added to the cis chamber. Spontaneous channel
activity was always tested for sensitivity to EGTA and Ca^2 +. The chamber to which
the addition of EGTA inhibited the activity of the incorporated channel presuma-
bly corresponds to the cytosolic side of the Ca^2 + release channel. The direction of
single channel currents was always measured from the luminal to the cytosolic side
of the channel, unless mentioned otherwise. Recordings were filtered at 2,500 Hz.
Data analyses were carried out using the pCLAMP 8.1 software package (Axon
Instruments). Free Ca^2 + concentrations were calculated using a computer program
that has previously been described^63.
Statistical analysis. Data are mean ± s.e.m., derived from independent samples
or independent experiments. All experiments were performed with at least five
biological replicates. The GraphPad Prism 8.1 software was used to test for dif-
ferences between groups. We used Student’s t-test (paired, two-tailed) or one-way
ANOVA with a Dunnett’s post hoc test. P < 0.05 was considered to be statistically
significant.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this paper.Data availability
Atomic coordinates and electron microscopy density maps of the following struc-
tures have been deposited in the PDB (http://www.rcsb.org) and the Electron
Microscopy Data Bank (EMDB https://www.ebi.ac.uk/pdbe/emdb/). FKBP12.6/
apo-CaM (PDB, 6JI8; EMDB, EMD-9833), FKBP12.6/ATP/caffeine/low-[Ca^2 +]/
CaM-M (PDB, 6JII; EMDB, EMD-9834), FKBP12.6/ATP/caffeine/low-[Ca^2 +]
(PDB, 6JI0; EMDB, EMD-9831), FKBP12.6/ATP/caffeine/low-[Ca^2 +]/Ca^2 +-CaM
(PDB, 6JIU; EMDB, EMD-9836), CHAPS- and DOPC-treated FKBP12.6/ATP/
caffeine/low-[Ca^2 +] (PDB, 6JRR; EMDB, EMD-9879), CHAPS- and DOPC-treated
FKBP12.6/ATP/caffeine/low-[Ca^2 +]/Ca^2 +-CaM (PDB, 6JRS; EMDB, EMD-9880),
FKBP12.6/ATP/caffeine/high-[Ca^2 +]/Ca^2 +-CaM (PDB, 6JIY; EMDB, EMD-9837)
and PCB95/low-[Ca^2 +]/Ca^2 +-CaM (PDB, 6JV2; EMDB: EMD-9889) complexes.
Source Data for Fig. 2e, f and Extended Data Figs. 1c, 6f, 7d–h are available in the
online version of the paper. All other data are available from the corresponding
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